1. Field of the Invention
The invention in the fields of microbiology and medicine relates to methods for rapid early detection of mycobacterial disease in humans based on the presence of antibodies to particular "early" mycobacterial antigens which have not been previously recognized for this purpose. Assay of such antibodies on select partially purified or purified mycobacterial preparations containing such early antigens permits diagnosis of TB earlier than has been heretofore possible. Also provided is a surrogate marker for screening populations at risk for TB, in particular subjects infected with human immunodeficiency virus (HIV).
2. Description of the Background Art
Recent estimates by the World Health Organization (WHO) suggest that approximately 90 million new cases of tuberculosis ("TB") will occur during this decade leading to about 30 million deaths (Raviglione, M. C. et al., 1995, JAMA. 273:220-226). The spread of HIV in populations already having a high incidence of TB related to socioeconomic factors and malnutrition has resulted in a resurgence of TB all over the world (Raviglione, M. C. et al., 1992, Bull World Health Organ 70:515-526; Harries A. D., 1990, Lancet. 335:387-390). This resurgence has renewed interest in developing improved vaccines, diagnostics, drugs and drug delivery regimens for TB. Furthermore, the immune dysfunction caused by HIV infection leads to a high rate of reactivation of latent TB, increased susceptibility to primary disease, as well as an accelerated course of disease progression (Raviglione et al., 1992, supra; 1995, supra; Shafer R. W. et al., 1996, Clin. Infect. Dis. 22:683-704; Barnes P. F. et al., 1991, N. Engl. J. Med. 324:1644-1650; Selwyn P. A. et al., 1989, N. Engl. J Med. 320:545-550).
It is well established that cellular immunity is critical for protection against TB. Much of the work in this field is focused on defining the antigens of the causative bacterium, Mycobacterium tuberculosis (M. tuberculosis; also abbreviated herein as "Mt") that can elicit effective immunity and on understanding the role of various cell populations in host-pathogen interactions (Andersen, P. et al., 1992, Scand. J. Immunol. 36:823-831; Havlir, D. V. et al., 1991, Infect. Immun. 59:665-670; Orme, I. M. et al., 1993, J. Infect. Dis. 167:1481-1497).
Delayed hypersensitivity measured as cutaneous immune reactivity to a purified protein derivative of Mt (abbreviated "PPD") is the only marker available for detection of latent infection with Mt. However, the sensitivity of the PPD skin test is substantially reduced during HIV infection (Raviglione et al., 1992, supra, 1995, supra; Graham N. M. H. et al., 1991, JAMA 267:369-373; Huebner R. E. et al., 1994, Clin. Infect. Dis. 19:26-32; Huebner R. E. et al., 1992, JAMA 267:409-410; Caiaffa W. T. et al., 1995, Arch. Intern. Med. 155:2111-2117). Furthermore, vaccination with a closely related mycobacterium designated Bacillus Calmette-Guerin (BCG) or previous exposure to other mycobacterial species can lead to false positive results in a PPD skin test. Not only does PPD reactivity fail to distinguish active, subclinical disease from latent infection, but the time between a positive skin test and development of clinical disease may range from months to several years (Selwyn P. A. et al., supra).
Because of the susceptibility of immunocompromised individuals to TB, the U.S. Centers for Disease Control and Prevention recommends preventive isoniazid therapy for all HIV seropositive (HIV.sup.+), PPD-positive (PPD.sup.+) individuals. However, the optimal time for such therapy is not clear and, ideally, should coincide with replication of previously latent bacteria. Unnecessary therapy must be minimized because prolonged isoniazid treatment can have serious toxic side effects (Shafer et al., supra). The impact of such treatment on emergence of drug resistant bacteria is still unclear. The use of preventive therapy in developing countries is seriously limited by the high frequency of PPD.sup.+ individuals coupled with the lack of adequate medico-social infrastructure and economic resources. High risk populations are also found in the United States, primarily intravenous drug users, homeless people, prison inmates and residents of slum areas (Fitzgerald, J. M. et al., 1991, Chest 100:191-200; Graham, N. M. H. et al., 1992, JAMA 267:369-373; Friedman, L. N. et al., 1996, New Engl. J. Med 334:828-833). Thus, discovery of additional surrogate markers for early detection and prompt treatment of active, subclinical TB in such high risk populations is urgently required.
Antibody responses in TB have been studied for several decades primarily for the purpose of developing serodiagnostic assays. Although some seroreactive antigens/epitopes have been identified, interest in antibody responses to M. tuberculosis has waned because of the lack of progress in simple detection of corresponding antibodies. Studies using crude antigen preparations revealed that healthy individuals possess antibodies that cross-react with several mycobacterial antigens. Such antibodies are believed to have been elicited by exposure to commensal and environmental bacteria and vaccinations (Bardana, E. J. et al., 1973, Clin. Exp. Immunol. 13:65-77; Das, S. et al., 1992, Clin. Exp. Immunol. 89:402-406; Del Giudice, G. et al., 1993, J. Immunol. 150:2025-2032; Grange, J. M., 1984, Adv. Tuberc. Res. 21:1-78; Havlir, D. V. et al., supra; Ivanyi, J. et al., 1989, Brit. Med. Bull. 44:635-649; Verbon, A. et al., 1990, J. Gen. Microbiol. 136:955-964). During the last decade, several mycobacterial antigens have been isolated and characterized (Young, D. B. et al., 1992, Mol. Microbiol. 6:133-145), including the 71 kDa DnaK, 65 kDa GroEL, 47 kDa elongation factor tu, 44 kDa PstA homologue, 40 kDa L-alanine dehydrogenase, 38 kDa PhoS, 23 kDa superoxide dismutase, 23 kDa outer membrane protein, 12 kDa thioredoxin, and the 14 kDa GroES. However, a majority of the antigens identified so far bear significant homology to the analogous proteins in other mycobacteria and non-mycobacterial prokaryotes (Andersen, A. B. et al., 1992, Infect. Immun. 60:2317-2323; Andersen, A. B. et al., 1989, Infect. Immun. 57:2481-2488; Braibant, M. et al., 1994, Infect. Immun. 62:849-854; Carlin, N. I. A. et al., 1992, Infect. Immun. 60:3136-3142; Garsia, R. J. et al., 1989, Infect. Immun. 57:204-212; Hirschfield, G. R. et al., 1990, J. Bacteriol. 172:1005-1013; Shinnick, T. M. et al., 1989, Nucl. Acids Res. 17:1254; Shinnick, T. M. et al., 1988, Infect. Immun. 56:446-451; Wieles, B. et al., 1995, Infect. Immun. 63:4946-4948; Young, D. B. et al., supra; Zhang, Y. et al., 1991, Mol. Microbiol. 5:381-391). Thus, almost all individuals (healthy or diseased) have antibodies to epitopes of conserved regions of these antigens. These antibodies are responsible for the uninformative (and possibly misleading) cross-reactivity observed with crude Mt antigen preparations (Davenport, M. P. et al., 1992, Infect. Immun. 60:1170-1177; Grandia, A. A. et al, 1991, Immunobiol. 182:127-134; Meeker, H. C. et al., 1989, Infect. Immun. 57:3689-3694; Thole, J. et al., 1987, Infect. Immun. 55:1466-1475).
Because such cross-reactive antibodies would mask the presence of antibodies specific for Mt antigens, some of the purified antigens such as the 38 kDa PhoS, the 30/31 kDa "antigen 85" (discussed in more detail below), 19 kDa lipoprotein, 14 kDa GroES and lipoarabinomannan have been prepared and tested (Daniel, T. et al., 1985 Chest. 88:388-392; Drowart, L. et al., 1991, Chest. 100:685-687; Jackett, P. S. et al., 1988, J. Clin. Microbiol. 26:2313-2318; Ma, Y. et al., 1986, Am. Rev. Respir. Dis. 134:1273-1275; Sada, E. et al., 1990, J. Clin. Microbiol. 28:2587-2590; Sada, E. D. et al., 1990, J. Infect. Dis. 162:928-931; Van Vooren, J. P. et al., 1991, J. Clin. Microbiol. 29:2348-2350). It is noteworthy that the choice of which antigen to test was dictated primarily by (a) its availability, (b) its immunodominance in animal immunizations, or (c) ease of its biochemical purification. None of these criteria take into account the reactivity of the antigen which occurs naturally in the human immune response to mycobacterial diseases. Use of the 38 kDa antigen has provided the highest serological sensitivity and specificity so far (Daniel, T. M. et al., 1987, Am. Rev. Respir. Dis. 135:1137-1151; Harboe, M. et al., 1992, J. Infect. Dis. 166:874-884; Ivanyi, J. et al., 1989, supra). However, in contrast to the present invention, the presence of anti-38 kDa antibodies is associated primarily with treated, advanced and recurrent TB (Bothamley, G. H. et al., 1992, Thorax. 47:270-275; Daniel, T. M. et al., 1986, Am. Rev. Respir. Dis. 134:662-665; Ma, Y. et al., 1986, Am. Rev. Respir. Dis. 134:1273-1275).
One convention in mycobacterial protein nomenclature is the use of MPB and MPT numbers. MPB denotes a protein purified from M. bovis BCG followed by a number denoting its relative mobility in 7.7% polyacrylamide gels at a pH of 9.5. MPT denotes a protein isolated from M. tuberculosis. In proteins examined prior to this invention, no differences in the N-terminal amino acid sequence were shown between these two mycobacterial species.
Wiker and colleagues have studied a family of secreted Mt proteins which include a complex of 3 proteins termed antigens 85A, 85B and 85C (also known as the "85 complex" or "85cx") (Wiker, H. G. et al., 1992, Scand. J. Immunol. 36:307-319; Wiker, H. G. et al., 1992, Microbiol. Rev. 56:648-661). This complex was originally found in M. bovis BCG preparations which produced a secreted antigen comprising a complex of three closely related components, antigen 85A, 85B, and 85C (Wiker, H. G. et al. 1986, Int. Arch. Allergy Appl. Immunol. 81:289-306). The corresponding components of Mt are also actively secreted. The 85 complex is considered the major secreted protein constituent of mycobacterial culture fluids though it is also found in association with the bacterial surface. In most SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, 85A and 85C are not properly resolved, whereas isoelectric focusing resolves three distinct bands.
Genes encoding six of the secreted proteins: 85A, 85B, 85C, "antigen 78" (usually referred to as the 38 kDa protein), MPB64 and MPB70 have been cloned . Three separate genes located at separate sites in the mycobacterial genome encode 85A, B and C (Content, J. et al., 1991, Infect. Immun. 59:3205-3212). A gene encoding the antigen known as MPT-32 (reported as a 45/47 kDa secreted antigen complex) has been cloned, sequenced and expressed (Laqueyrerie, A. et al., 1995, Infec. Immun. 63:4003-4010) and designated as the apa gene. One of the present co-inventors and his collaborators provided evidence for glycosylation sites on this protein (Dobos, K. M. et al., 1996, J. Bacteriol. 178:2498-2506 and Example III herein). However, the need continues for further elucidation of the biochemistry and inmmunochemistry of Mt proteins and glycoproteins which are potentially important as serodiagnostic tools. The full definition of glycosylation sites and the nature and extent of glycosylation of glycosylated proteins has been scant. Initial evidence for the presence of glycoproteins in Mt was based on the observation of discrete concanavalin A (ConA)-binding products upon PAGE and electroblotting of protein preparations. However, since these patterns occurred in the midst of considerable quantities of mannose (Man)-containing lipoglycans and phospholipids (Dobos et al., supra), chemical proof of amino acid glycosylation is still considered necessary and is provided as part of this invention.
The antigen 85 complex is often referred to as the "30/31 kDa doublet," although slightly different molecular mass designations have been reported. The following list shows the molecular masses of the individual components of antigen 85 complex plus two additional antigens (in SDS-PAGE) as described by Wiker and colleagues, along with alternative nomenclatures:
Ag85A = MPT44 = 31 kDa Ag85B = MPT59 = 30 kDa Ag85C = MPT45 = 31.5 kDa MPT64 = 26 kDa MPT51 = 27 kDa Ag78 -- = 38 kDa MPT32 = 45/47 kDa (found to be 38/42 kDa by the present inventors)
Wiker's group studied cross-reactions between five actively secreted Mt proteins by crossed immunoelectrophoresis, SDS-PAGE with immunoblotting and enzyme immunoassay (EIA) using (1) polyclonal rabbit antisera to the purified proteins and (2) a mouse monoclonal antibody ("mAb"). The mAb HBT4 reacted with the MPT51 protein. The 85A, 85B, and 85C constituents cross-reacted extensively, though each had component-specific in addition to cross-reacting epitopes. These components also cross-reacted with MPT51 and MPT64. Amino acid sequence homology was shown between 85A, 85B, 85C and MPT51. MPT64 showed less homology. Striking homology was also found between two different structures within the 85B sequence. Thus a family of at least four secreted proteins with common structural features has been demonstrated in mycobacteria. Three of these proteins bind readily to fibronectin (Abou-Zeid, C., 1988, Infect. Immun. 56:3046-3051; Abou-Zeid, C., 1988, Infect. Immun. 59:2712-2718; Harboe, M. et al., 1992, Clin. Inf. Dis. 14:313-319).
The aligned amino acid sequences listed below illustrate the homology of a fragment of 85A, 85B, 85C, MPT51 and MPT64. The numbers at the top correspond to the part of the sequence shown. The N-terminal sequences were determined on isolated proteins and aligned by visual inspection. The sequence from position 66 to 91 of MPT64 is the sequence deduced from the cloned gene.
 SEQ 1 5 10 15 20 25 30 35 ID NO 85A(1-39) FSRPGLPVEYLQVPS PSMGRDIKVQFQSGGANSP ALYLL 1 85B(1-39) FSRPGLPVEYLQVPS PSMGRDIKVQFQSGGNNSP AVYLL 2 85C(1-37) FSRPGLPVEYLQVPSA SMGRDIKVQFQGGG PHAVYLL 3 MPT51(1-32) APYENLMYPS PSMGRDKPVAFLAGG PHAVYLL 4 MPT64(66-91) APYE LNITSATYQS AIPPRG TQAVVL 5
The N-terminal sequence of MPT51 showed 72% homology with the sequence of the Ag 85 components (when P at position 2 is aligned with P at position 7 of the three Ag 85 components.
Apart from fibronectin binding, little information concerning the primary functions of antigen 85 complex proteins is available. Although the art has not considered antibodies as playing a significant role in protective immunity against mycobacterial infections, Wiker et al. (supra) speculated that the existence of interactions between Ag 85 and fibronectin implied that an antibody to Ag 85 which could block this interaction might affect early events in disease progression and increase host resistance.
Studies of TB patients showed that assays of antibodies to the Ag 85 complex had a sensitivity of about 50%. With regard to specificity, the Ag 85 components are highly cross-reactive so that positive responses are expected (and found) in healthy controls, particularly in geographic areas of high exposure to atypical mycobacteria The different degree of specificity is thus highly dependent on the kind of control subjects used. It is noteworthy that traditional BCG vaccination does not appear to induce a significant antibody response, though it is interesting that antibodies to mycobacterial antigens increased when anti-TB chemotherapy was initiated.
C. Espitia et al., 1989, Clin Exp Immunol 77:373-377, found antibodies to the 30/31 kDa doublet band (presumably 85A and 85C) in 55.9% of TB patient sera (and in 56.5% of lepromatous leprosy sera). Sera from healthy individuals often showed binding which was weaker than TB patients. Van Vooren, J. P. et al., 1991, J. Clin. Microbiol. 29:2348-2350, found that antigen 85A reacted with sera from tuberculous as well as nontuberculous individuals. By contrast, 85B and 85C did not react with the control sera but reacted with 20 of 28 serum samples (71%) from tuberculous patients. Wiker and colleagues concluded that the future of the serology of antibody responses to antigen 85 would require investigation of antibodies to component-specific epitopes and in particular to species-specific epitopes. The extensive cross-reactivity of antigen 85 in different species of mycobacteria suggested to Wiker et al. (supra) that tests could attain sufficient sensitivity, though suitable mAbs were said to be essential for further development of tests for infection with Mt (and atypical mycobacteria). Importantly, the present inventors note the deficiency in the art of analysis of antibodies at different stages of disease. This is one of the primary deficiencies addressed by this invention.
C. Espitia et al., 1995, Infect. Immun. 63:580-584, found reciprocal cross-reactivity between a Mt 50/55 kDa protein and a M. bovis BCG 45/47 kDa antigen using a rabbit polyclonal antiserum against the M. bovis protein and a mAb against the Mt antigen. Both antigens were secreted glycoproteins. The N-terminal sequences and total amino acid content of these proteins were very similar. Analysis by 2D gel electrophoresis showed at least seven different components in the Mt 50/55 kDa antigen. In solid-phase immunoassays, purified Mt 50/55 kDa protein was recognized by sera from 70% of individuals (n=77) with pulmonary TB. The N-terminus of the Mt 41 kDa antigen known as MPT32 was very similar to the N-termini of the 50/55 kDa--and the 4547 kDa proteins. The molecular mass of this Mt protein was deduced to be 45-47 kDa. Espitia et al., supra, speculated about a diagnostic potential for these antigens based on their observation of antibodies in 70% of their TB patients. However, the potential of this antigen as an early diagnostic agent for TB was neither analyzed nor even suggested.
In sum, none of the antigens studied so far, with the possible exception of MPT32 (as will be described herein) has emerged as a suitable candidate for development of a diagnostic assay for early stages of TB. Since antigens/epitopes recognized during natural infection and disease progression in humans may differ substantially from those recognized by animals upon artificial immunization (Bothamley, G. et al., 1988, Eur. J. Clin. Microbiol. Infect. Dis. 7:639-645; Calle, J. et al., 1992, J. Immunol. 149:2695-2701; Hartskeerl, R. A. et al., 1990, Infect. Immun. 58:2821-2827; Laal, S. et al., 1991, Proc. Natl. Acad Sci. USA. 88:1054-1058; Meeker, H. C. et al., 1989, Infect. Immun. 57:3689-3694; Verbon, A., 1994, Trop. Geog. Med. 46:275-279), there is a pressing need in the art for selection of antigens based on their ability to stimulate the human immune system. This would permit the identification of such useful antigens and design of diagnostic assays for early detection of TB.
TB in HIV Infected Subjects
Studies aimed at determining the integrity of humoral immune memory during HIV infection have shown that the ability to respond to recall antigens by producing significant amounts of high-affinity specific IgG antibodies was maintained during the time prior to onset of clinical AIDS (Janoff, E. N. et al., 1991, J. Immunol. 147:2130-2135). Secondary antibody responses are relatively independent of T cell help, and B cells specific for recall antigens are present in normal frequency in HIV-infected individuals (Janoff et al. (supra); Kroon F. P. et al., 1995, Clin. Infect. Dis. 21:1197-1203). Comparison of secondary responses to different antigens in HIV-infected individuals also suggested that the level of immunologic memory established prior to HIV infection may influence the ability of the subject to respond post-infection (Janoff et al. (supra)). Since TB in HIV-infected individuals often results from reactivation of latent infection, and reactivated TB is known to occur relatively early during the course of HIV disease progression, the immune system may be sufficiently intact to generate antibody responses towards bacteria emerging from latency. If this occurs, HIV-infected subjects with active TB infection should have detectable antibodies directed towards Mt antigens.
Although the literature on TB infection in subjects not infected with HIV is extensive, reports on antibody responses of HIV/TB patients to M. tuberculosis, have been scant and controversial. Farber, C. et al., 1990, J. Infect. Dis, 162:279-280, reported the presence of antibodies to the p32 antigen (same as 85A) in 7 of 8 HIV/TB patients. Da Costa, C. et a., 1993, Clin. Exp. Immunol. 91:25-29, reported the presence of anti-lipoarabinomannan (LAM) antibodies in 35% of such patients. Barer, L. et al., 1992, Tuber. Lung. Dis. 73:187-191, reported anti-PPD antibodies in 36% of HIV/TB patients. Martin-Casabona, N. et al, 1992, J. Clin. Microbiol. 30:1089-1093, reported anti-sulfolipid (SLIV) antibodies in 73% of their patients. In addition, van Vooren, P. et al., 1988, Tubercle. 69:303-305, reported that anti-p32 antibodies were detectable in an HIV/TB patient for several months prior to clinical manifestation of TB. In contrast, analysis of responses to Ag60 (Saltini C. et al., 1993, Am. Rev. Respir. Dis. 145:1409-1414; van der Werf, T. S. et al., 1992. Med Microbiol Immunol 181:71-76) and Ag85B (McDonough, J. A. et al., 1992, J. Lab. Clin. Med. 120:318-322) failed to detect antibodies in these patients.
Hence, there is a particular need in the art for methods to detect TB infections at early stages in HIV patients since they comprise one of the largest populations at risk for TB throughout the world.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.